Fundamentals II: Bacterial Genetics

1. Fundamentals II:Bacterial Genetics Janet Yother, Ph.D. Department of Microbiology jyother@uab.edu 4-9531

2. Learning Objectives • Bacterial transcription and translation • Examples of transcriptional regulation • Gene transfer mechanisms • Roles of mutation, gene transfer, and recombination in virulence and antibiotic resistance

3. transcription translation DNA (m)RNA protein reverse transcription (some viruses) replication replication Central Dogma of Molecular Biology

4. Cytoplasmic membrane d.s. circular single s.c. RNA polymerase - recognizes specific sequences (promoters) in DNA to initiate transcription s.s. linear NH2-(aa)n-COOH Replication - DNA polymerase and other enzymes Ribosome - recognizes specific sequences (Ribosome binding sites) in mRNA to initiate translation; catalyzes amino acid additions transcription translation DNA mRNA protein

5. Promoters and Transcription Initiation in Bacteria Molecular Genetics of Bacteria 2nd Ed, 2003

6. mRNA 3’ UCAGUCGUG 5’ mRNA 5’ AGUCAGCAC 3’ http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/ chroms-genes-prots/transcription-translation.html Transcription(DNA mRNA) DNA 5’ AGTCAGCAC 3’ 3’ TCAGTCGTG 5’ mRNA - synthesized 5’ to 3’ - complement of DNA (U instead of T)

7. Transcription can occur on either DNA strand - the one used depends on the presence of the proper signals On average, 1 gene ~ 1 kb Escherichia coli ~ 4500 kb Streptococcus pneumoniae ~ 2300 kb

8. Cytoplasmic membrane d.s. circular single s.c RNA polymerase - recognizes specific sequences (promoters) in DNA to initiate transcription s.s. linear Replication - DNA polymerase and other enzymes Ribosome - recognizes specific sequences (Ribosome binding sites) in mRNA to initiate translation; catalyzes amino acid additions Transcription/Translation in Bacteria transcription translation DNA mRNA protein

9. Translation(mRNA polypeptide) • Initiation - 30S subunit of ribosome binds mRNA at specific site • 30S subunit binds 50S subunit 70S • Synthesis - tRNA anticodons pair with complementary codons in mRNA, add amino acid to growing chain

10. 3’ end of 16S rRNA 3’ 5’ A N U N UCCUCCA 5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’ mRNA Shine- Delgarno sequence Initiation Codon Translation Initiation Ribosome (30S) Ribosome Binding Site

11. aminoacyl-tRNA to A-site of ribosome peptidyl transfer (peptide bind formation) translocation Molecular Biology of the Cell 4th Ed, 2002

12. Translation - Genetic Code • Essentially universal • Amino acid determined by mRNA codon (codon = 3 nucleotides; complement of anticodon in tRNA) • Translation start = AUG (Met); less often GUG • Translation stop = UAA, UAG, UGA • Exception: UGA in mycoplasma = Trp

13. ribosome subunits protein Simultaneous translation of same mRNA by multiple ribosomes http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/chroms-genes-prots/transcription-translation.html

14. RNAP DNA mRNA AUG ribosome growing polypeptide Science 169: 392-395. Coupled Transcription-Translation

15. Operons Bacterial genes can be organized into operons - more than one gene transcribed from a single promoter Promoter = non-coding sequence RBS RBS RBS RBS mRNA monocistronic message mRNA polycistronic message No introns in bacteria although there is non-coding sequence

16. Mechanisms of Transcriptional Regulation • Alternative Sigma Factors • Two component (signal transduction) • Quorum sensing

17. Alternative Sigma Factors • Bind RNA polymerase, allow recognition of alternative promoter sequences • s70 (70 kDa) = major sigma factor in E. coli • At least 8 alternative sigma factors • s54 – nitrogen limitation • s32 – heat shock • Bacillus – sporulation

18. Signal Transduction • Two-component regulatory systems. Input signal Sensor autophosphorylates (usually histidine kinase) ) P ATP Interacts with and phosphorylates RR Response regulator - Mediates downstream effects P Signals include - temperature, O2, phosphate, sugar Downstream effects include DNA binding and transcription alterations, protein interactions

19. Quorum Sensing • Accumulation and detection of small molecule leads to transcription regulation • Gram-negative signal = acyl homoserine lactone • Gram-positive signal = oligopeptide Gram-positive Gram-negative ABC- transporter Two-comp Regulator

20. Mutations • Any change in DNA sequence whether effect observable or not • Causes • spontaneous - errors in DNA replication. Arise at a low but constant and often detectable frequency (always occurring). • induced - radiation (X-ray, uv), chemicals. Increase frequency.

21. Classes and Results of Mutations - I • Point Mutation • alteration of single nucleotide. Can have multiple point mutations. • Possible Results • samesense - codes for same amino acid. No effect (silent). • missense - codes for different amino acid. Protein function may/may not be altered. • nonsense - now codes for translation stop codon. Premature stop >> truncated product, function probably lost (depending on where stop occurs)

22. X DNA mRNA RBS RBS Polar effect of insertion - multiple genes may be affected due to transcription from same promoter Classes and Results of Mutations - II • Deletion - DNA lost. Function lost if most/all of gene deleted. • Insertion - new DNA has been added. Gene interrupted. Function usually lost.

23. Recombination - Homologous • Occurs between regions of DNA that are highly similar • Involves specific bacterial enzymes • RecA-mediated

24. Recombination: Non-homologous • Occurs between DNAs without significant similarity. Best example, important in pathogens - transposons. • Transposons - mobile (transposable) genetic elements (Tn) • DNA sequences that can insert essentially at random into chromosome/plasmid (some have some site specificities) • result is an insertion mutation: disrupt function, polar (affect expression of downstream genes) • 2 to 50 kb • cannot replicate autonomously • encode functions for own transposition • often, encode antibiotic resistance (Amp, Km, e.g.), virulence factors.

25. Bacterial Gene Transfer Mechanisms

26. Extrachromosomal DNA • Plasmids - Replicate in cytoplasm, independent of chromosome. • Usually circular (borrelia = linear) • Few to several hundred kb • Few to several hundred copies • Conjugative (F, R), antibiotic resistance, metabolic, virulence • Bacteriophage - virus; replicates in cytoplasm or integrates into chromosome • Seen with electron microscope • DNA or RNA; no metabolic apparatus • Specific phage infects specific bacterium(a)

27. Bacteriophage • virus; replicates in cytoplasm or integrates into chromosome • Seen with electron microscope • DNA or RNA (in phage head); no metabolic apparatus • Specific phage infects specific bacterium(a) • Types • Virulent - continually in lytic cycle, making phage; bacterial host usually killed • Temperate - may undergo lytic cycle OR lysogenic cycle (symbiotic with host; may encode virulence factors); >90% of known phages

28. Significance of bacteriophage • Phage (lysogenic) conversion - observable effect of phage carried by bacterium. Medically important. Every bacterium may carry a phage. • Corynebacterium diptheriae - Gm + rod; diptheria toxin = phage-encoded • Clostridium botulinum - Gm + rod; botulism toxin = phage-encoded • Gene transfer (transduction)

29. Transduction • Mediated by bacteriophage • Transduction = accidental packaging of bacterial DNA during lytic cycle, transfer to new host (transducing phages) 100s released

30. Conjugation F-pilus • Mediated by F-factor or similar conjugative plasmids (in Gram-negatives) • F-factors can encode antibiotic resistance = R factor • F-factors replicate in cytoplasm and be transferred - - - F ~ 100 kb Donor F+ Recipient F- one strand of F transferred; replicated in donor and recip Both donor and recipient = F+

31. Conjugation F-pilus • OR F-factors can integrate into chromosome and transfer part of the chromosome enters last enters first Genes near oriT transferred most frequently F rarely transferred - recipient does not become Hfr • Mediated by pheromones in Gram-positives

32. Transformation • Uptake and integration into chromosome (usually) of free DNA (plasmids can also be transformed) • First demonstrated in Streptococcus pneumoniae (1928)

33. Transformation • Homologous DNA integrated (though non-homologous DNA may be taken up by some bacteria) • DNA from lysed bacteria or secretion • Highly regulated - uptake machinery may be expressed only when other like bacteria are present • Gm +: Streptococcus, Bacillus, Streptomyces • Gm -: Haemophilus, Pseudomonas, Neisseria

34. Roles of Mutation, Recombination, and Gene Transfer in Virulence and Antibiotic Resistance

35. Variation - Antigenic • Antigenic Variation (Microbial evasion) • Antigenic drift - slow accumulation of point or other “small” mutations. Alter specific protein at one or few antigenic epitopes (influenza virus) • Antigenic shift - major change. Results from recombination (new DNA from gene transfer; intracellular deletions, insertions) • Permanent change

36. Variation - Phase • Phase Variation (Microbial Variation) • Switching back and forth between expressing/not expressing • Involves recombination • Not permanent, can revert to original type • Advantages for Pathogen • Avoid antibody, avoid having antibody made to antigen • Express antigen only when important (attachment, e.g.)

37. Variation - Phase On/off of one antigen: E. coli pili involved in attachment. Inversion of promoter. Expression of alternative antigens Neisseria gonorrhoeae pili

38. Genetics and Antibiotic Resistance

39. Antibiotics - Mechanisms of Action (Differences between Prokaryotes and Eukaryotes) • inhibit protein synthesis • bind ribosomal proteins, RNA polymerase • Aminoglycosides (kanamycin), tetracyclines, macrolides (erythromycin) • Rifampin (binds RNA polymerase) • inhibit DNA synthesis • bind enzymes/proteins involved in DNA replication • Fluoroquinolones (ciprofloxacin) • inhibit metabolic activity • bind enzymes • Sulfonamides-bactrim (inhibit tetrahydrofolate production) • Cell wall synthesis • Penicillins (block transpeptidation) • Vancomycin (blocks transglycosylation)

40. Antibiotics - Specificity for Bacteria • Differences between bacterial (70S) and mammalian ribosomes (80S) • Analogous mammalian enzymes insensitive • Antibiotic doesn’t enter mammalian cells • Absence of peptidoglycan in mammalian cells

41. Antibiotic Mechanism Inhibit DNA replication (gyrase) Inhibit transcription (RNA polymerase) Inhibit translation (ribosome) Inhibit Cell Wall Synthesis Bacterial Resistance Altered gyrase doesn’t bind antibiotic (point mutations*) Altered RNA polymerase (point mutations*) Altered ribosome*; enzyme (plasmid-encoded) inactivates antibiotic; protein (plasmid-encoded) prevents antibiotic entry into cell Altered cell wall synthesis proteins*; enzyme (plasmid-encoded) inactivates antibiotic * chromosome-encoded

42. Development and Spread of Antibiotic Resistance • Mutations (point, in chromosome) • Plasmids, transposons • Gene transfer • **Mutations arise at a low but constant frequency. • **Antibiotics SELECT FOR naturally-occurring resistant isolates.